Next-generation Sequencing Findings Research Articles

The Johns Hopkins Molecular Tumor Board Precision Oncology elective for Medical Oncology fellows.

11035 Background: The accelerated impact of next generation sequencing (NGS) in clinical decision making requires the integration of cancer genomics and precision oncology focused training into medical oncology education. The Johns Hopkins Molecular Tumor Board (JH MTB) is a multi-disciplinary effort focused on integration of NGS findings with critical evidence interpretation to generate personalized recommendations tailored to the genetic footprint of individual patients. Methods: The JH MTB and the Medical Oncology Fellowship Program have developed a 3-month precision oncology elective for fellows in their research years. Commencing fall of 2020, the goals of this elective are to enhance the understanding of NGS platforms and findings, advance the interpretation and characterization of molecular assay outputs by use of mutation annotators and knowledgebases and ultimately master the art of matching NGS findings with available therapies. Fellow integration into the MTB focuses on mentored case-based learning in mutation characterization and ranking by levels of evidence for actionability, with culmination in form of verbal presentations and written summary reports of final MTB recommendations. A mixed methods questionnaire was administered to evaluate progress since elective initiation. Results: Three learners who have participated as of February 2021 were included. Of the two who had completed the MTB elective, each have presented at least 10 cases, with at least 1 scholarly publication planned. All indicated strong agreement that MTB elective had increased their comfort with interpreting clinical NGS reports as well as the use of knowledgebases and variant annotators. Exposure to experts in the field of molecular precision oncology, identification of resources necessary to interpret clinical NGS reports, development of ability to critically assess various NGS platforms, and gained familiarity with computational analyses relevant to clinical decision making were noted as strengths of the MTB elective. Areas of improvement included ongoing initiatives that involve streamlining variant annotation and transcription of information for written reports. Conclusions: A longitudinal elective in the JHU MTB has been found to be preliminarily effective in promoting knowledge mastery and creating academic opportunities related to the clinical application of precision medicine. Future directions will include leveraging of the MTB infrastructure for research projects, learner integration into computational laboratory meetings, and expansion of the MTB curriculum to include different levels of learners from multiple medical education programs. Continued elective participation will be key to understanding how best to facilitate adaptive expertise in assigning clinical relevance to genomic findings, ultimately improving precision medicine delivery in patient care and trial development.

Novel findings of splenic extramedullary hematopoiesis during primary myelofibrosis, post-essential thrombocythemia, and post-polycythemia vera myelofibrosis.

BCR-ABL-fusion-negative myeloproliferative neoplasms (MPNs) with myelofibrosis (MF) include primary MF, post-polycythemia vera MF and post-essential thrombocythemia MF. Clonal extramedullary hematopoiesis (EMH) can occur during MPN pathogenesis. Although histopathological bone-marrow (BM) features during clonal EMH have been investigated, those of the spleen have been poorly described. We analyzed splenectomy samples from 28 patients with MF and BM samples from 20 of them. Slides were stained with hematoxylin and eosin, reticulin, and trichrome, with immunohistochemical labeling of glycophorin A, myeloperoxidase, CD61, CD34, and CD117. We also subjected splenectomy and BM samples from six patients and spleen samples from seven patients to next-generation sequencing (NGS). Megakaryocyte-rich spleen nodules (MRSNs), seen in seven of the 28 patients, were significantly associated with megakaryocyte proliferation in the spleen (p = 0.04). We devised a grading system for spleen fibrosis (SF) and found that SF was increased in 20 of 28 patients. Notably, patients with SF were more likely to have MRSNs, suggesting that megakaryocytes might participate in SF, as previously described in BM. Comparisons of spleen and BM NGS findings of six patients' specimens revealed identical mutational status in the two organs for half of the patients. We observed additional mutations in the spleen of two patients. However, the meaning of this finding remains unknown since there was a long interval between BM and spleen samplings (68 and 82months, respectively).

Detection of Pathogenic Bacteria From Septic Patients Using 16S Ribosomal RNA Gene-Targeted Metagenomic Sequencing.

Conventional blood cultures were compared to plasma cell-free DNA-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR)/next-generation sequencing (NGS) for detection and identification of potential pathogens in patients with sepsis. Plasma was prospectively collected from 60 adult patients with sepsis presenting to the Mayo Clinic (Minnesota) Emergency Department from March through August 2019. Results of routine clinical blood cultures were compared to those of 16S rRNA gene NGS. Nineteen (32%) subjects had positive blood cultures, of which 13 yielded gram-negative bacilli, 5 gram-positive cocci, and 1 both gram-negative bacilli and gram-positive cocci. 16S rRNA gene NGS findings were concordant in 11. For the remaining 8, 16S rRNA gene NGS results yielded discordant detections (n = 5) or were negative (n = 3). Interestingly, Clostridium species were additionally detected by 16S rRNA gene NGS in 3 of the 6 subjects with gastrointestinal sources of gram-negative bacteremia and none of the 3 subjects with urinary sources of gram-negative bacteremia. In the 41 remaining subjects, 16S rRNA gene NGS detected at least 1 potentially pathogenic organism in 17. In 15, the detected microorganism clinically correlated with the patient's syndrome. In 17 subjects with a clinically defined infectious syndrome, neither test was positive; in the remaining 7 subjects, a noninfectious cause of clinical presentation was identified. 16S rRNA gene NGS may be useful for detecting bacteria in plasma of septic patients. In some cases of gram-negative sepsis, it may be possible to pinpoint a gastrointestinal or urinary source of sepsis based on the profile of bacteria detected in plasma.

Evaluating Infectious, Neoplastic, Immunological, and Degenerative Diseases of the Central Nervous System with Cerebrospinal Fluid-Based Next-Generation Sequencing.

Cerebrospinal fluid (CSF) is a clear and paucicellular fluid that circulates within the ventricular system and the subarachnoid space of the central nervous system (CNS), and diverse CNS disorders can impact its composition, volume, and flow. As conventional CSF testing suffers from suboptimal sensitivity, this review aimed to evaluate the role of next-generation sequencing (NGS) in the work-up of infectious, neoplastic, neuroimmunological, and neurodegenerative CNS diseases. Metagenomic NGS showed improved sensitivity—compared to traditional methods—to detect bacterial, viral, parasitic, and fungal infections, while the overall performance was maximized in some studies when all diagnostic modalities were used. In patients with primary CNS cancer, NGS findings in the CSF were largely concordant with the molecular signatures derived from tissue-based molecular analysis; of interest, additional mutations were identified in the CSF in some glioma studies, reflecting intratumoral heterogeneity. In patients with metastasis to the CNS, NGS facilitated diagnosis, prognosis, therapeutic management, and monitoring, exhibiting higher sensitivity than neuroimaging, cytology, and plasma-based molecular analysis. Although evidence is still rudimentary, NGS could enhance the diagnosis and pathogenetic understanding of multiple sclerosis in addition to Alzheimer and Parkinson disease. To conclude, NGS has shown potential to aid the research, facilitate the diagnostic approach, and improve the management outcomes of all the aforementioned CNS diseases. However, to establish its role in clinical practice, the clinical validity and utility of each NGS protocol should be determined. Lastly, as most evidence has been derived from small and retrospective studies, results from randomized control trials could be of significant value.

Novel RB1 and MET Gene Mutations in a Case with Bilateral Retinoblastoma Followed by Multiple Metastatic Osteosarcoma.

Retinoblastoma (Rb) is a malignant tumor of the developing retina that affects children before the age of five years in association with inherited or early germline mutations of the RB1 gene. The genetic predisposition is also a driver for other primary malignancies, which have become the leading cause of death in retinoblastoma survivors. Other malignancies can occur as a consequence of radiotherapy. We describe a patient with retinoblastoma in which we detected a novel RB1 c.2548C > T, p.(Gln850Ter) and a synchronous MET c.3029C > T, p.(Thr1010Ile) mutation as well. After presenting with bilateral retinoblastoma, the patient developed at least four different manifestations of two independent osteosarcomas. Our goal was to identify all germline and somatic genetic alterations in available tissue samples from different time periods and to reconstruct their clonal relations using next generation sequencing (NGS). We also used structural and functional prediction of the mutant RB and MET proteins to find interactions between the defected proteins with potential causative role in the development of this unique form of retinoblastoma. Both histopathology and NGS findings supported the independent nature of a chondroblastic osteosarcoma of the irradiated facial bone followed by an osteoblastic sarcoma of the leg (tibia).

Culture-independent Next Generation Sequencing of Urine and Expressed Prostatic Secretions in Men With Chronic Pelvic Pain Syndrome

To compare standard cultures and next-generation sequencing (NGS) in men with chronic prostatitis/chronic pelvic pain syndrome (CPPS). CPPS shares clinical features with urinary tract infections, but bacteria are seldom found. NGS is more sensitive than standard cultures. Men diagnosed with CPPS (National Institute of Health Category III) underwent traditional cultures and NGS of their urine and expressed prostatic secretions (EPS). Characteristics between groups were compared statistically. Thirty-one men with CPPS were included (mean age 44.5). All standard urine cultures were negative, and 3 EPS cultures were positive. Seventy-eight unique microbes were detected with NGS, including uropathogens in 10 of the men. There were no bacteria identified by NGS in EPS that were not also found in the urine. Men with positive NGS did not differ from those without in age, symptom severity or phenotype. Men with typical urinary tract infection symptoms (eg, dysuria, chills) were more likely to have uropathogens detected on NGS relative to men without such symptoms. Nine patients were prescribed antibiotics based on their NGS findings, but only 1 exhibited symptom improvement (11%). NGS commonly identified bacteria in CPPS patients, but these did not localize to the prostate. NGS positivity did not correlate with symptom severity and antibiotic therapy was seldom effective. NGS detected uropathogens more frequently in those with clinical symptoms suggestive of urinary tract infection. Clinical trials are needed to examine the utility of NGS-guided antibiotics in this subpopulation.

Next-generation assessment of human epidermal growth factor receptor 2 gene (ERBB2) amplification status in invasive breast carcinoma: a focus on Group 4 by use of the 2018 American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the testing guideline in 2018 to address issues arising from uncommon human epidermal growth factor receptor 2 (HER2) fluorescence in-situ hybridisation (FISH) results according to the 2013 guideline. Next-generation sequencing (NGS) may be used to better classify patients. The aim of this study was to assess the ERBB2 amplification status of invasive breast carcinoma with equivocal HER2 immunohistochemistry (IHC) results by using NGS, focusing on Group 4 (HER2/CEP17 ratio of <2.0; average HER2 signals/cell of ≥4.0 and <6.0). We retrospectively reviewed HER2 FISH and NGS data of HER2 IHC-equivocal breast carcinomas at our centre between January 2009 and September 2019, wherein all three assays were performed on the same tissue block, and compared HER2 FISH results, according to the 2018 ASCO/CAP guideline, and the ERBB2 amplification status determined with NGS. A total of 52 HER2 FISH and NGS results from 51 patients with HER2 IHC-equivocal breast carcinomas were reviewed. The cohort included eight cases classified as 2018 ASCO/CAP in-situ hybridisation Group 1, three classified as Group 2, three classified as Group 3, 14 classified as Group 4, and 24 classified as Group 5. Thirteen of 14 (92.9%) Group 4 (HER2-negative) cases were classified as ERBB2-non-amplified by the use of NGS; the discordant case was later classified as Group 1 with alternative sample FISH testing. NGS revealed no significant difference in somatic mutations or copy number alterations between Groups 4 and 5. Our NGS findings support the reclassification of HER2 FISH-equivocal cases as HER2-negative under the 2018 ASCO/CAP guideline.

Genomic study of dilated cardiomyopathy in a group of Mexican patients using site-directed next generation sequencing.

BackgroundDilated cardiomyopathy (DCM) is a major cause of nonischemic heart failure and death in young adults. Next generation sequencing (NGS) has become part of the diagnostic workup in idiopathic and familial DCM. More than 50 DCM genes have been identified, revealing great molecular heterogeneity and variable diagnostic yield. Interpretation of variant pathogenicity is challenging particularly in underrepresented populations, as pathogenic variant databases include studies mainly from European/Caucasian populations. To date, no studies on genomic diagnosis of DCM have been conducted in Mexico.MethodsWe recruited 55 unrelated DCM patients, 22 familial (F‐DCM), and 33 idiopathic (I‐DCM), and performed site‐directed NGS seeking causal mutations. Diagnostic yield was defined as the proportion of individuals with at least one pathogenic (P) or likely pathogenic (LP) variant in DCM genes.ResultsOverall diagnostic yield was 47.3%, and higher in F‐DCM (63.6%) than in I‐DCM (36.4%, p = 0.047). Overall, NGS disclosed 41 variants of clinical interest (61.0% novel), 27 were classified as P/LP and 14 of unknown clinical significance. Of P/LP variants, 10 were A‐band region TTN truncating variants, five were found in DSP (18.5%), five in MYH7 (18.5%), two in LMNA (7.4%), and one in RBM20, ABCC9, FKTN, ACTA1, and TNNT2. NGS findings suggested autosomal recessive inheritance in three families, two with DSP loss of function mutations in affected individuals. The increasing number of mutation reports in DCM, increasing knowledge on the functional consequences of mutations, mutational hotspots and functional domains of DCM‐related proteins, the recent refinement ACMG/ClinGen Guidelines, and co‐segregation analysis in DCM families helped increase the diagnostic yield.ConclusionThis is the first NGS study performed in a group of Mexican DCM patients, contributing to understand the mutational spectrum and complexity of DCM molecular diagnosis.

Reduced sensitivity for EGFR T790M mutations using the Idylla EGFR Mutation Test

AimsOsimertinib is a third-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor that is effective in non-small cell lung cancer (NSCLC) harbouring the EGFR T790M mutation. The Idylla EGFR Mutation...

Molecular profiling of metastatic breast cancer (MBC) and target-based therapeutic matching in an Asian tertiary phase I oncology unit.

3561 Background: Somatic profiling of MBC has highlighted actionable mutations and driven trials of matched targeted therapy (tx). Previous phase I studies have reported improved outcomes following matched therapies with tumour molecular profiles. Here, we review next generation sequencing (NGS) and treatment outcomes of Asian MBC patients (pts) in the phase I unit of a tertiary centre. Methods: Pts with MBC referred to a phase I unit underwent NGS (n = 152). Tumour tissue was sequenced via the amplicon based Ion Ampliseq Cancer (IAC) v2 (50 genes) platform from 2014-2017 prior to institutional change to Foundation Medicine 1 (FM1) (324 genes) 2017-2019. Patients were counselled on findings and enrolled onto matched therapeutic trials where available. Results: NGS was successfully performed in 107 pts (IAC 46%, FM1 54%) of which tumour subtypes include hormone receptor positive 63%, triple negative breast cancer (TNBC) 28% and Her2 positive 19%. Median lines of prior tx for MBC was 4 (range 0-12). 89% had prior chemotherapy (CT), 57% prior endocrine therapy (ET). 72/107 (67%) sequenced patients had further treatment and 18 (25%) were matched to tx based on NGS findings (15 clinical trial, 3 off trial). Matching rates on both NGS platforms were similar (IAC 22% vs FM1 28%). Mutated pathways with potential matched tx included PIK3CA/AKT/PTEN (52%), DNA damage response (DRR) (15%), and FGFR (11%) pathways. PIK3 mutations were seen in 43% and associated with higher number of metastatic sites (p = 0.03); most prevalent aberrations were PIK3CA H1047R (41%) and PIK3CA E542K (13%). Matched cases were more heavily pretreated (mean lines of prior tx 5.3 matched vs 3.7, unmatched p = 0.05), and showed a median progression free survival (mPFS) of 24 weeks [w] and clinical benefit rate (complete/partial response or stable disease ≥ 12 weeks) of 53% on matched tx. Comparison by NGS platform showed improved mPFS for matched vs unmatched pts sequenced on FM1 vs IAC (FM1: 26 vs 19w, HR = 0.76 [95% CI: 0.3-1.9]; IAC: 8 vs 12w; HR = 1.21 [95% CI: 0.5-2.8]). Interestingly, 1 pt with SMARCB mutation, reportedly associated with the FGFR pathway, had a PFS of 70w on tx with a pan-FGFR inhibitor after progressing on 3 prior lines of tx (ET and CT). Conclusions: Molecular profiling of MBC in a phase I unit led to matched tx in 25% of cases. Matched pts showed encouraging mPFS with a suggestion of benefit in those matched after sequencing on a broader gene panel (FM1).

Pulmonary microbiome patterns correlate with the course of disease in patients with sepsis-induced ARDS following major abdominal surgery

Patients with sepsis-induced Acute Respiratory Distress Syndrome (ARDS) are hallmarked by high mortality rates. Early, targeted antibiotic therapy is crucial for patients' survival. The clinical use of a Next Generation Sequencing (NGS)-based approach for pathogen identification may lead to an improved diagnostic performance. Therefore, the objective of this study was to examine changes in the pulmonary-microbiome and resulting influences on patients' outcome in septic ARDS, but also to compare NGS- and culture-based diagnostic methods for pathogen identification. In total, 30 patients in two groups were enrolled in the study: (1) 15 septic ARDS patients following major abdominal surgery and (2) 15 patients undergoing oesophageal resection serving as controls. In the ARDS group, blood samples were collected at ARDS onset as well as 5 days and 10 days afterwards. At the same timepoints, bronchoalveolar lavages (BAL) were performed to collect epithelial lining fluid for culture-, as well as NGS-based analyses and to evaluate longitudinal changes in the pulmonary microbiome. In the control group, only one BAL and one blood sample were collected. ARDS patients showed a significantly reduced α-diversity (p=0.007**) and an increased dominance (p=0.012*) in their pulmonary-microbiome. The α-diversity-index correlated with the length of stay in the intensive care unit (p-value=0.015) and the need for mechanical ventilation (p-value=0.009). In 42.9% of all ARDS patients, culture-based results were negative, while NGS findings indicated bacterial colonization. Sepsis-induced ARDS is associated with a significant dysbiosis of patients' pulmonary-microbiome, which is closely correlated with the clinical course of the disease. This prospective, observational pilot study was approved by the Ethics Committee of the Medical Faculty of Heidelberg (trial code no. S-063/2015) and was prospectively registered in the German clinical trials register (DRKS-ID: DRKS00008317 prospectively registered: 28.10.2015). All study patients or their legal representatives signed written informed consent.

Multiplexed molecular profiling of lung cancer with malignant pleural effusion using next generation sequencing in Chinese patients.

Lung cancer is the most common type of cancer and the leading cause of cancer-associated death worldwide. Malignant pleural effusion (MPE), which is observed in ~50% of advanced non-small cell lung cancer (NSCLC) cases, and most frequently in lung adenocarcinoma, is a common complication of stage III–IV NSCLC, and it can be used to predict a poor prognosis. In the present study, multiple oncogene mutations were detected, including 17 genes closely associated with initiation of advanced lung cancer, in 108 MPE samples using next generation sequencing (NGS). The NGS data of the present study had broader coverage, deeper sequencing depth and higher capture efficiency compared with NGS findings of previous studies on MPE. In the present study, using NGS, it was demonstrated that 93 patients (86%) harbored EGFR mutations and 62 patients possessed mutations in EGFR exons 18–21, which are targets of available treatment agents. EGFR L858R and exon 19 indel mutations were the most frequently observed alterations, with frequencies of 31 and 25%, respectively. In 1 patient, an EGFR amplification was identified and 6 patients possessed a T790M mutation. ALK + EML4 gene fusions were identified in 6 patients, a ROS1 + CD74 gene fusion was detected in 1 patient and 10 patients possessed a BIM (also known as BCL2L11) 2,903-bp intron deletion. In 4 patients, significant KRAS mutations (G12D, G12S, G13C and A146T) were observed, which are associated with resistance to afatinib, icotinib, erlotinib and gefitinib. There were 83 patients with ERBB2 mutations, but only two of these mutations were targets of available treatments. The results of the present study indicate that MPE is a reliable specimen for NGS based detection of somatic mutations.

Challenges in reporting pathogenic/potentially pathogenic variants in 94 cancer predisposing genes - in pediatric patients screened with NGS panels

The benefit of reporting unsolicited findings in Next Generation Sequencing (NGS) related to cancer genes in children may have implications for family members, nevertheless, could also cause distress. We aimed to retrospectively investigate germline variants in 94 genes implicated in oncogenesis, in patients referred to NGS testing for various rare genetic diseases and reevaluate the utility of reporting different classes of pathogenicity. We used in silico prediction software to classify variants and conducted manual review to examine unsolicited findings frequencies in 145 children with rare diseases, that underwent sequencing - using a 4813 gene panel. The anonymized reanalysis revealed 18250 variants, of which 126 were considered after filtering. Six pathogenic variants (in BRCA1,BMPR1A,FANCA,FANCC,NBN genes) with cancer related phenotype and three unsolicited variants (in BRCA2,PALB2,RAD50 genes) were reported to patients. Additionally, three unsolicited variants in ATR, BLM (in two individuals), and FANCB genes presented potential cancer susceptibility, were not reported to patients. In retrospect, 4.8% (7/145) of individuals in our cohort had unsolicited NGS findings related to cancer. More efforts are needed to create an updatable consensus in reporting variants in cancer predisposing genes, especially for children. Consent process is crucial to inform of both value and risk of additional genetic information.

Clinical Impact of Next-generation Sequencing in Pediatric Neuro-Oncology Patients: A Single-institutional Experience.

The implementation of next-generation sequencing (NGS) in pediatric neuro-oncology may impact diagnosis, prognosis, therapeutic strategies, clinical trial enrollment, and germline risk. We retrospectively analyzed 58 neuro-oncology patients (31 boys, 27 girls, average age 7.4 years) who underwent NGS tumor profiling using a single commercially available platform on paraffin-embedded tissue obtained at diagnosis (20 low-grade gliomas, 12 high-grade gliomas, 11 embryonal tumors, four ependymal tumors, three meningeal tumors, and eight other CNS tumors) from May 2014 to December 2016. NGS results were analyzed for actionable mutations, variants of unknown significance and clinical impact. Seventy-four percent of patients (43 of 57) had actionable mutations; 26% had only variants of uncertain significance (VUS). NGS findings impacted treatment decisions in 55% of patients; 24% were given a targeted treatment based on NGS findings. Seven of eight patients with low-grade tumors treated with targeted therapy (everolimus, trametinib, or vemurafenib) experienced partial response or stable disease. All high-grade tumors had progressive disease on targeted therapy. Forty percent of patients had a revision or refinement of their diagnosis, and nine percent of patients were diagnosed with a previously unconfirmed cancer predisposition syndrome. Turnaround time between sample shipment and report generation averaged 13.4 ± 6.4 days. One sample failed due to insufficient DNA quantity. Our experience highlights the feasibility and clinical utility of NGS in the management of pediatric neuro-oncology patients. Future prospective clinical trials using NGS are needed to establish efficacy.

T69MICROBIOME PROFILES ARE ASSOCIATED WITH COGNITIVE FUNCTIONING IN 45-MONTH-OLD CHILDREN

Patients with sepsis-induced acute respiratory distress syndrome (ARDS) have a high mortality rate. Early, targeted antibiotic therapy is crucial for patient survival. The clinical use of a next-generation sequencing (NGS)-based approach for pathogen identification may lead to improved diagnostic performance. Therefore, the objectives of this study were to examine changes in the pulmonary microbiome and resulting influences on patient outcome in sepsis-induced ARDS, and to compare NGS- and culture-based diagnostic methods for pathogen identification.In total, 30 patients in two groups were enrolled in the study: 15 patients with sepsis-induced ARDS following major abdominal surgery and 15 patients undergoing oesophageal resection (serving as controls). In the ARDS group, blood samples were collected at ARDS onset (day 0), and 5 and 10 days later. Bronchoalveolar lavage (BAL) was performed at the same timepoints to collect epithelial lining fluid for culture- and NGS-based analyses, and to evaluate longitudinal changes in the pulmonary microbiome. In the control group, only one BAL and one blood sample were collected.Patients with ARDS showed significantly lower α-diversity (P=0.007) and increased dominance (P=0.012) in their pulmonary microbiome compared with the controls. The α-diversity index correlated with the length of stay in the intensive care unit (P=0.015) and the need for mechanical ventilation (P=0.009). In 42.9% of patients with ARDS, culture-based results were negative while NGS findings indicated bacterial colonization.Sepsis-induced ARDS is associated with significant dysbiosis of the patient's pulmonary microbiome, which is closely correlated with the clinical course of disease.

Abstract 2412: Development of a gene signature to predict positive or negative sentinel nodes as a prognostic marker in melanoma

Abstract In recent years, sentinel lymph node (SLN) biopsy has been adopted as a less invasive technique to assess the prognostic outcome of melanoma, whereby draining the key lymph nodes for presence of a tumor decreases unnecessary lymph node dissections and reduces the risk of developing lymphedema. However, this technique only has therapeutic value in patients with SLN-positive tumor nodes, which only represents about half of the population diagnosed with melanoma. In a pilot study, we assessed the predictive efficacy of using a commercialized multi-biomarker next-generation sequencing (NGS) assay (Illumina® Comprehensive Cancer Panel) containing selected genes which have been shown to have a prognostic impact in melanoma progression, to determine whether these can identify primary tumors which do not require SLN biopsy. We used laser capture microdissection to capture pathologist-identified areas of tumor from four groups of samples 1) skin lesions from SLN-positive patients, 2) skin lesions from SLN-negative patients, 3) node samples from SLN-positive patients and node samples from SLN-negative patients; some cases of skin lesions and nodes were matched for the same patient. Sequencing identified 14 genes containing variants in &amp;gt;70% of the positive node samples, which were absent in samples with negative nodes. This included EGFR, PDGFR, ATM, PIK3CA, TP53 and ERBB4. Of note, variants in ERBB4 is of interest as it has been shown in the past to be a potential molecular target in the treatment of melanoma. We are currently validating these preliminary findings in a larger cohort of patients (n=31) and will report on the NGS findings of this study and the efficacy of using genetic biomarkers in skin biopsy samples as a potential prognostic marker for early stage melanoma patients who may not require SLN biopsy. Citation Format: Audrey J. Majeske, Timothy J. Geddes, Richard Keidan, George D. Wilson. Development of a gene signature to predict positive or negative sentinel nodes as a prognostic marker in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2412.

Identification of Clonal Hematopoiesis Mutations in Solid Tumor Patients Undergoing Unpaired Next-Generation Sequencing Assays.

In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays. This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing. Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis. Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care.See related commentary by Pollyea, p. 5790.

Clinical Utility of Multiple-Gene Mutation Testing By Next-Generation Sequencing in Acute Myeloid Leukemia in Community Hospital Setting

Clinical Utility of Multiple-Gene Mutation Testing by Next-Generation Sequencing in Acute Myeloid Leukemia in Community Hospital SettingIntroductionThe WHO 2016 classification system and the 2017 European LeukemiaNet (ELN) genetic risk stratification system incorporate cytogenetic aberrations and mutations of specific genes to classify acute myeloid leukemia (AML) and stratify prognosis of AML patients. Next-generation sequencing (NGS) of multiple genes associated with AML has been shown to be an important tool in studying AML in research and academic center settings. Our goal is to evaluate the clinical application of NGS in a community hospital setting which has not been well documented leading to rejection of coverage by some insurers.MethodologyThis retrospective study included 109 patients (52 females and 57 males with mean age of 58.7) from November, 2016 to June, 2018 with various races/ethnicities background and confirmed diagnosis of AML at Florida Hospital (FH). DNA was extracted from the peripheral blood/bone marrow for NGS analysis. A total of 54 hematopoietic tumor associated genes were studied: ABL1, ASXL1, ATM, BCOR, BIRC3, BRAF, CALR, CBL, CEBPA, CREBBP, CSF1R, CSF3R, DNMT3A, EP300, ETV6, EZH2, FBXW7, FGFR4, FLT3, GATA1, GATA2, GATA3, IDH1, IDH2, IL7R, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MPL, NF1, NOTCH1, NOTCH2, NPM1, NRAS, NSD1, PAX5, PDGFRA, PDGFRB, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TERT, TET1, TET2, TP53, TSLP,U2AF1 and ZRSR2. The sequencing was performed at Genomics and Pathology Services laboratory at Washington University in St. Louis and initial informatics analysis was performed by PierianDX using Clinical Genome Working platform. The initial analysis results were delivered to the pathologists at FH to do final interpretation with integration of the results of morphology/flow cytometry/karyotyping studies.The clinical utility of NGS was evaluated by: 1) comparing the AML classification and ELN genetic risk stratification of each patient with and without NGS data, 2) identifying gene mutations for targeted therapy and 3) other clinical impacts.ResultsNGS results changed ELN risk stratification in 45 cases (41.3%) and WHO AML classification in 37 cases (33.9%) out of 109 cases after considering the mutation status of NPM1, FLT3, RUNX1, ASXL1 and TP53. Among the 45 cases that had changes of ELN risk, 25 cases (55%) were downgraded to favorable risk group all because of mutations in NPM1 gene. While, the rest 20 cases (45%) were upgraded to adverse risk group due to the following mutations (RUNX1 in 7, TP53 in 5, ASXL1 in 4, RUNX1 and ASXL1 in 4). The change of WHO classification was caused by mutations in NPM1 (n=26) or RUNX1 (n=11). Additionally, NGS identified 8 cases with either GATA2 or CEBPA gene mutation, which are considered to increase the risk of myeloid neoplasms with germline predisposition based on WHO classification. Finally, NGS identified 42 patients (38.5%) carrying FLT3-ITD (n=17), FLT3-TKD (n=8) and/or IDH1/IDH2 (n=16) mutations. These mutations are known to have approved targeted therapies (FLT3 inhibitors and IDH2 inhibitors) or ongoing clinical trials (IDH1 inhibitors). Overall, NGS results led to clinically significant impacts in 62/109 (56.9%) patients by changing the AML classification/risk stratification, finding potential germline mutation and/or targeted therapies.DiscussionOur results indicate that NGS findings provide important information for classification, risk stratification, genetic predisposition, and treatment options in the majority (62/109, 56.9%) of AML patients in a community hospital setting. How the information exactly changes the clinical management by community practicing hematologists is currently ongoing and will provide further insights in regards to the clinical utilities of NGS. In addition, NGS of multiple genes is more cost- and labor-effective than single gene sequencing. Taken together, the insurers should accept NGS testing as a standard of care for AML patients. DisclosuresNo relevant conflicts of interest to declare.

Towards an External Quality Assessment for Next Generation Sequencing in the Diagnosis of Rare Inherited Anaemias

The diagnosis of patients with inherited anemias is increasingly made in conjunction with high-throughput ‘next-generation’ sequencing (NGS) analysis, largely using targeted resequencing panels validated for clinical use in diagnostic labs. While there is a joint UK NEQAS and European Molecular Quality Network pilot scheme for Molecular Genetics to assess NGS quality control, this is not disease-specific. Patients with inherited anaemias can have multiple mutations with complex genotype phenotype interactions therefore a scheme assessing interpretation of these results could be of value. Likewise, guidelines for variant reporting (eg ACMG- American College of Medical Genetics and Genomics) provide excellent advice on how to interpret the likely pathogenicity of genetic variants, but no disease-specific guidance exists to assist in the clinical interpretation of NGS findings for individuals with rare inherited anemias. The European Hematology Association Scientific Working Group on Red Cells and Iron carried out a survey among its members to investigate variability in current practice and determine the need for, and feasibility of, a formal external quality assessment (EQA) scheme. Surveys and two clinical vignettes with sample variant call files (VCFs) were distributed among 14 participating labs from 9 countries; 13/14 labs used a targeted panel and 1/14 lab (8%) used a 366-gene virtual panel derived from whole exome sequencing data. Accreditation: 12/14 labs had ISO (International Organisation for Standardisation) accreditation for their NGS; 2/12,used local accreditation schemes. The number of genes per targeted panel ranged from 18 to 215 genes (median 64), covering: congenital dyserythropoietic anemias, Diamond-Blackfan anemia, sideroblastic anemia, red cell membrane and enzyme disorders. Some panels included other related bone marrow failure or iron metabolism disorders. 7/13 labs with targeted panels sequenced only exons, with variable padding into introns, while 6/13 routinely sequenced 3‘ and 5‘ untranslated regions. Capture methods were variable between labs and 11/14 labs used Illumina platforms for sequencing and 3/14 Ion Torrent. Sanger sequencing was used for confirmation of NGS variants in 12/14 labs, but used for gap-filling of uncovered regions in 10/14 labs. Reporting of variants followed ACMG guidelines in 10/14 labs, ACGS (Association for Clinical Genomics Science) guidelines in 2/14 and no published guidelines in 2/14. Reporting of Tier 3 (variants of uncertain significance): 8/14 labs reported strict adherence to ACMG guidelines, including only Class 4 and Class 5 variants in clinical reports, while 6/14 labs admitted looser adherence and reporting of Class 3 variants depending on circumstances. The number of samples analysed per year was highly variable between labs (10-600, median 60). Two mini-EQA VCFs were sent with clinical vignettes, for which 100% of labs correctly identified a case of autosomal recessive sideroblastic anaemia due to compound heterozygous mutations in SLC25A38, and 100% correctly identified a case negative by NGS. Class 3 variants were not reported at all in 50% of clinical reports, reported in the main body of the report in 20% and in a separate table in 30% of labs. In conclusion, we have identified common approaches to NGS sequencing in 14 diagnostic laboratories but highlighted variability in accreditation, use of Sanger sequencing and adherence to ACMG guidelines. The feasibility of carrying out an EQA scheme has been established and work will continue with UK NEQAS to formally create such a scheme, with the aim of ensuring improved patient care through the use of objective quality assessments. DisclosuresColombatti:ADDMEDICA: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; NOVARTIS: Consultancy. Viprakasit:Protagonist Therapeutics: Consultancy, Research Funding; Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Clinical Utility of Genomic Profiling in the Treatment of Advanced Sarcomas: A Single-Center Experience.

Sarcomas are a diverse group of malignant tumors that arise from soft tissues or bone. For most advanced cases, there is a substantial need for improved therapeutic options and, therefore, a desire to more precisely tailor therapy in individual cases. In this study, we review our institutional experience with next-generation sequencing (NGS)-based molecular profiling for non-GI stromal tumors sarcomas, with a focus on the clinical utility of the results. We retrospectively analyzed results of NGS performed on tumors from 114 patients with a diagnosis of sarcoma. A chart review was conducted to review the clinical impact of NGS findings. A median of three putatively oncogenic gene alterations were identified per tumor sample (range, 0 to 19) and at least one mutation was detected in 96.7% of tumors. Fifty-six patients (49.1%) harbored a finding that was felt to be actionable after review by a molecular tumor board. Five patients (4.4%) had a diagnosis change as a result of NGS findings. In 15 patients (13.2%), therapeutic selection was influenced by NGS findings. Four of 15 (26.7%) of the NGS-influenced systemic therapies resulted in clinical benefit. Putatively oncogenic mutations are readily detected in the majority of sarcomas. Genetic profiling affected the diagnosis and/or treatment approach in a sizeable minority of patients with sarcoma treated at our center. Additional study is required to determine if genetic profiling leads to improved clinical outcomes.